Human peptidyl-prolyl isomerases

ABSTRACT

The invention provides human peptidyl-prolyl isomerases (HPPIP) and polynucleotides which identify and encode HPPIP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of HPPIP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of humanpeptidyl-prolyl isomerases and to the use of these sequences in thediagnosis, treatment, and prevention of cancer andautoimmune/inflammatory disorders.

BACKGROUND OF THE INVENTION

Numerous essential biochemical reactions involve the isomerization of asubstrate. Enzymes which catalyze such reactions are known asisomerases. A number of isomerases have been described catalyzing stepsin a wide variety of biochemical pathways including protein folding,phototransduction, and various anabolic and catabolic pathways (e.g.,glycolysis), in organisms ranging from bacteria to human.

One class of isomerases is known as peptidyl-prolyl cis-trans isomerases(PPIases). PPIases catalyze the cis to trans isomerization of certainproline imidic bonds in proteins. Two families of PPIases are thecyclophilins (CyPs), and the FK506 binding proteins (FKBPs). CyP wascharacterized originally as the receptor for the immunosuppressant drugcyclosporin, an inhibitor of T-cell activation. Subsequent workdemonstrated that CyPs isomerase activity is essential for correctprotein folding. Thus, the peptidyl-prolyl isomerase activity of CyP maybe part of the signaling pathway that leads to T-cell activation.(Bergsma, D. J. et al (1991) J. Biol. Chem. 266:23204-23214.)

FKBPs bind the potent immunosuppressants FK506 and rapamycin, therebyinhibiting signaling pathways in T-cells. Specifically, the PPIaseactivity of FKBPs is inhibited by binding FK506 or rapamycin. There arefive members of the FKBP family which are named according to theircalculated molecular masses (FKBP12, FKBP13, FKBP25, FKBP52, andFKBP65), and are localized to different regions of the cell where theyassociate with different protein complexes. FKBP12 is localized to thecytoplasm and is associated with the ryanodine receptor and the inositol1,4,5-trisphosphate receptor. FKBP13 is located in the endoplasmicreticulum where it's PPIase activity assists in folding growingpolypeptide chains. FKBP25 is found in the nucleus and associates withnucleolin and casein kinase II. FKBP52 associates with unactivatedsteroid receptors. FKBP65 has been localized to the membrane, but noproteins have yet been shown to interact with it. (Coss, M. et al.(1995) J. Biol. Chem. 270:29336-29341; Schreiber, S. L. (1991) Science251:283-287.)

Other isomerases are involved in essential biochemical reactionpathways. For example, in E. coli, 3,4-dihydroxyphenylacetate isconverted to succinic semialdehyde in an aromatic catabolism pathwayknown as the homoprotocatechuate pathway. This pathway requires twoisomerization steps. The first step is the conversion of5-carboxymethyl-2-hydroxymuconic acid to5-oxo-pent-3-ene-1,2,5-tricarboxylic acid by the action of5-carboxymethyl-2-hydroxymuconate isomerase. In the second step, theenzyme 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase (HHDDI) catalyzesthe isomerization of 2-hydroxy-hepta-2,4-diene-1,7-dioic acid into2-oxo-hepta-3-ene-1,7-dioic acid. These isomerization steps areessential to the breakdown of aromatic compounds which producessubstrates for energy metabolism. (Roper, D. I. and Cooper, R. A. (1993)Eur. J. Biochem. 217: 575-580.)

The discovery of new human peptidyl-prolyl isomerases and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, treatment, andprevention of cancer and autoimmune/inflammatory disorders.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides, humanpeptidyl-prolyl isomerases, referred to collectively as “HPPIP” andindividually as; “HPPIP-1” and “HPPIP-2.” In one aspect, the inventionprovides a substantially purified polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.

The invention further provides a substantially purified variant havingat least 90% amino acid identity to the amino acid sequences of SEQ IDNO: 1 or SEQ ID NO:2, or to a fragment of either of these sequences. Theinvention also provides an isolated and purified polynucleotide encodingthe polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2. The invention also includes anisolated and purified polynucleotide variant having at least 90%polynucleotide seqeunce identity to the polynucleotide encoding thepolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and afragment of SEQ ID NO:2.

Additionally, the invention provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:2, as wellas an isolated and purified polynucleotide having a sequence which iscomplementary to th(polynucleotide encoding the polypeptide comprisingthe amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ IDNO:2.

The invention also provides an isolated and purified polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3, and a fragmentof SEQ ID NO:4. The invention further provides an isolated and purifiedpolynucleotide variant having at least 90% polynucleotide sequenceidentity to the polynucleotide sequence comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4, as well as anisolated and purified polynucleotide having a sequence which iscomplementary to the polynucleotide comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, afragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO:2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:2.In another aspect, the expression vector is contained within a hostcell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO: 1, and a fragment ofSEQ ID NO:2, the method comprising the steps of: (a) culturing the hostcell containing an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide under conditions suitable forthe expression of the polypeptide; and (b) recovering the polypeptidefrom the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, afragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2 in conjunctionwith a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO: 1, anda fragment of SEQ II) NO:2, as well as a purified agonist and a purifiedantagonist to the polypeptide. The invention also provides a method fortreating or preventing a cancer, the method comprising administering toa subject in need of such treatment an effective amount of an antagonistof the polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO: 1, anda fragment of SEQ ID NO:2. In addition, the invention provides a methodfor treating an autoimmune/inflammatory disorder, the method comprisingadministering to a subject in need of such treatment an effective amountof an antagonist of the polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, afragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.

The invention also provides a method for detecting a polynucleotideencoding the polypeptide comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQID NO:1, and a fragment of SEQ ID NO:2 in a biological sample containingnucleic acids, the method comprising the steps of: (a) hybridizing thecomplement of the polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO: 1, and a fragment ofSEQ ID NO:2 to at least one of the nucleic acids of the biologicalsample, thereby forming a hybridization complex; and (b) detecting thehybridization complex, wherein the presence of the hybridization complexcorrelates with the presence of a polynucleotide encoding thepolypeptide in the biological sample. In one aspect, the nucleic acidsof the biological sample are amplified by the polymerase chain reactionprior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:3) of HPPIP-1. The alignment wasproduced using MacDNASIS PRO™ software (Hitachi Software Engineering Co.Ltd., San Bruno, Calif.).

FIGS. 2A, 2B, 2C, and 2D show the amino acid sequence (SEQ ID NO:2) andnucleic acid sequence (SEQ ID NO:4) of HPPIP-2. The alignment wasproduced using MacDNASIS PRO™ software.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended (claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

DEFINITIONS

“HPPIP,” as used herein, refers to the amino acid sequences; ofsubstantially purified HPPIP obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, whenbound to HPPIP, increases or prolongs the duration of the effect ofHPPIP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of HPPIP.

An “allelic variant,” as this term is used herein, is an alternativeform of the gene encoding HPPIP. Allelic variants may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

“Altered” nucleic acid sequences encoding HPPIP, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same as HPPIPor a polypeptide with at least one functional characteristic of HPPIP.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HPPIP, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding HPPIP. The encodedprotein may also be “altered,” and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent HPPIP. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of HPPIP is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments,” “immunogenic fragments,” or“antigenic fragments” refer to fragments of HPPIP which are preferablyabout 5 to about 15 amino acids in length, most preferably 14 aminoacids, and which retain some biological activity or immunologicalactivity of HPPIP. Where “amino acid sequence” is recited herein torefer to an amino acid sequence of a naturally occurring proteinmolecule, “amino acid sequence” and like terms are not meant to limitthe amino acid sequence to the complete native amino acid sequenceassociated with the recited protein molecule.

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp. 1-5.)

The term “antagonist,” as it is used herein, refers to a molecule which,when bound to HPPIP, decreases the amount or the duration of the effectof the biological or immunological activity of HPPIP. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HPPIP.

As used herein, the term “antibody” refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindHPPIP polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the “sense”strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation “negative” canrefer to the antisense strand, and the designation “positive” can referto the sense strand.

As used herein, the term “biologically active,” refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic HPPIP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides by base pairing. For example, thesequence “A-G-T” binds to the complementary sequence “T-C-A.”Complementarity between two single-stranded molecules may be “partial,”such that only some of the nucleic acids bind, or it may be “complete,”such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding HPPIP orfragments of HPPIP may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts, e.g., NaCl,detergents, e.g., sodium dodecyl sulfate (SDS), and other components,e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

“Consensus sequence,” as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW™ Fragment Assemblysystem (CCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HPPIP, byNorthern analysis is indicative of the presence of nucleic acidsencoding HPPIP in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding HPPIP.

A “deletion,” as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term “similarity,” as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word “identity” may substitute for the word “similarity.” Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as “substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MegAlign™ program (DNASTAR, Inc.,Madison Wis.). The MegAlign™ program can create alignments between twoor more sequences according to different methods, e.g., the clustalmethod. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no similarity between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be counted orcalculated by other methods known in the art, e.g., the Jotun Heinmethod. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)Identity between sequences can also be determined by other methods knownin the art, e.g., by varying hybridization conditions.

“Human artificial chromosomes” (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet, 15:345-355.)

The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

“Hybridization,” as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term “hybridization complex” refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀t or R₀t analysis) or formed between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray,” as used herein, refers to an arrangement ofdistinct polynucleotides arrayed on a substrate, e.g., paper, nylon orany other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

The terms “element” or “array element” as used herein in ;i microarraycontext, refer to hybridizable polynucleotides arranged on the surfaceof a substrate.

The term “modulate,” as it appears herein, refers to a change in theactivity of HPPIP. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HPPIP.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to a nucleotide, oligonucleotide, polynucleotide, or any fragmentthereof. These phrases also refer to DNA or RNA of genomic or syntheticorigin which may be single-stranded or double-stranded and may representthe sense or the antisense strand, to peptide nucleic acid (PNA), or toany DNA-like or RNA-like material. In this context, “fragments” refersto those nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can b(econtiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

The term “oligonucleotide,” as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

“Peptide nucleic acid” (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term “sample,” as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding HPPIP,or fragments thereof, or HPPIP itself, may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms “specific binding” or “specifically binding”refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between polynucleotides and the claimedpolynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent (e.g., formamide),temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

The term “substantially purified,” as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “variant” of HPPIP, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” (changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE™ software.

THE INVENTION

The invention is based on the discovery of new human peptidyl-prolylisomerases (HPPIP), the polynucleotides encoding HPPIP, and the use ofthese compositions for the diagnosis, treatment, or prevention of cancerand autoimmune/inflammatory disorders.

Nucleic acids encoding the HPPIP-1, corresponding to SEQ ID NO:3 of thepresent invention, were first identified in Incyte Clone 2291164 fromthe peripheral blood mononuclear cell cDNA library (TMLR3DT01) using acomputer search, e.g., BLAST, for amino acid sequence alignments.

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,1D, and 1E. HPPIP-1 is 472 amino acids in length and has acyclophilin-type peptidyl-prolyl cis-trans isomerase signature sequencefrom residue Y₄₉ through residue G₆₆. In addition, HPPIP-1 has twopotential N-glycosylation sites at residues N₁₀₉ and N₂₀₁, two potentialcAMP- and cGMP-dependent protein kinase phosphorylation sites atresidues T₂₉₃ and T₃₉₇, twelve potential casein kinase IIphosphorylation sites at residues T₂₉, S₇₃, S₁₄₅, S₂₀₆, S₂₃₁, S₂₉₉,S₃₁₁, S₃₄₆, T₃₇₉, S₃₈₀, T₄₁₁, and S₄₃₇, six potential protein kinase Cphosphorylation sites at residues S₂₂₄, S₂₅₀, S₃₇₅, T₃₇₉, S₄₃₀, andS₄₆₁, and a potential tyrosine kinase phosphorylation site at residueY₁₃₉. PFAM analysis identifies HPPIP-1 as a peptidyl-prolyl cis-transisomerase (pro_isomerase), with the region from residue N₁₂ throughresidue F₁₆₈ receiving a score of 150 bits. BLOCKS analysis alsoidentifies HPPIP-1 as a cyclophilin-type peptidyl-prolyl cis-transisomerase (BL00170), which the algorithm defines using three regionsdesignated BL00170A, BL00170B, and BL00170C. The region from residue R₉₆through residue N₁₄₀, matching region BL00170C, received a score of 1366on a strength of 1662, and is supported by the presence of regionsBL00170A and BL00170B with a P value less than 6.7×10 ⁻⁵. PRINTSanalysis also identifies HPPIP-1 as a cyclophilin peptidyl-prolylcis-trans isomerase (PR00153), which the algorithm defines using fiveregions designated PR00153A through PR00153E. The region from residueF₅₄ through residue G₆₆, matching region PR00153B, received a score of1475 on a strength of 1471, and is supported by the presence of regionsPR00153A, PR00153C, PR00153D, and PR00153E with a P value less than2.3×10⁻¹⁵. Northern analysis shows the expression of this sequence invarious libraries, at least 65% of which are immortalized or cancerousand at least 28% of which involve immune response. In addition, 28% ofthe libraries expressing HPPIP-1 are from reproductive tissue, and 21%are from nervous tissues. Of particular note is the expression ofHPPIP-1 in tumors of the breast, prostate, and brain.

Nucleic acids encoding the HPPIP-2 of the present invention were firstidentified in Incyte Clone 289973 from the normalized brain cDNA library(BRAINON01) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:4, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 289973H1 (BRAINON01), 1437423F1 (PANCNOT08), 1819932F6(GBLATUT01), 30 1920956H1 (BRSTTUT01), and 1996837R6 (BRSTTUT03).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:2, as shown in FIGS. 2A, 2B, 2C,and 2D. HPPIP-2 is 447 amino acids in length and has a potentialN-glycosylation site at residue N58, and a potential amidation site atresidue T₄₃₀. In addition, HPPIP-2 has two potential cAMP- andcGMP-dependent protein kinase phosphorylation sites at residues S₄₅, andT₂₅₁, seven potential casein kinase II phosphorylation sites at residuesS₃, S₆₀, T₁₅₂, T₁₆₆, S₂₂₅, S₂₅₀, and S₄₀₇, seven potential proteinkinase C phosphorylation sites at residues S₆₀, T₁₆₆, T₁₇₅, S₁₈₁, S₂₀₁,S₄₁₂, and T₄₃₀, and a potential tyrosine kinase phosphorylation site atresidue Y_(158.) BLOCKS analysis identifies HPPIP-2 as An FKBP-typepeptidyl-prolyl cis-trans isomerase (BL00453), which the algorithmdefines using three regions designated BL00453A, BL00453B, and BL00453C.The region from residue I₂₀₈ through residue L₂₂₁, matching regionBL00453C, received a score of 1071 on a strength of 1275. Northernanalysis shows the expression of this sequence in various libraries, atleast 53% of which are immortalized or cancerous and at least 44% ofwhich involve immune response. In addition, 25% of the librariesexpressing HPPIP-2 are from reproductive tissue, and 24% are fromgastrointestinal tissues. Of particular note is the expression ofHPPIP-2 in tumors of the breast, prostate, and brain.

The invention also encompasses HPPIP variants. A preferred HPPIP variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe HPPIP amino acid sequence, and which contains at least onefunctional or structural characteristic of HPPIP.

The invention also encompasses polynucleotides which encode HPPIP. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:3, which encodes HPPIP-1.In a further embodiment, the invention encompasses the polynucleotidesequence comprising the sequence of SEQ ID NO:4, which encodes HPPIP-2.

The invention also encompasses a variant of a polynucleotide sequenceencoding HPPIP. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding HPPIP. A particular aspect of theinvention encompasses a variant of SEQ ID NO:3 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:3. The inventionfurther encompasses a polynucleotide variant of SEQ ID NO:4 having atleast about 80%, more preferably at least about 90%, and most preferablyat least about 95% polynucleotide sequence identity to thepolynucleotide sequence, encoding HPPIP. Any one of the polynucleotidevariants described above can encode an amino acid sequence whichcontains at least one functional or structural characteristic of HPPIP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HPPIP, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet generic code as applied tothe polynucleotide sequence of naturally occurring HPPIP, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HPPIP and it; variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HPPIP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HPPIP or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding HPPIP and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeHPPIP and HPPIP derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding HPPIP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:3, SEQ ID NO:4, a fragment ofSEQ ID NO:3, or a fragment of SEQ ID NO:4, under various conditions ofstringency. (See, e.g., Wahl, G. M. and S. L,. Berger (1987) MethodsEnzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol.152:507-511.) For example, stringent salt concentration will ordinarilybe less than about 750 mM NaCl and 75 mM trisodium citrate, preferablyless than about 500 mM NaCl and 50 mM trisodium citrate, and mostpreferably less than about 250 mM NaCl and 25 mM trisodium citrate. Lowstringency hybridization can be obtained in the absence of organicsolvent, e.g., formamide, while high stringency hybridization can beobtained in the presence of at least about 35% formamide, and mostpreferably at least about 50% formamide. Stringent temperatureconditions will ordinarily include temperatures of at least about 30°C., more preferably of at least about 37° C., and most preferably of atleast about 42° C. Varying additional parameters, such as hybridizationtime, the concentration of detergent, e.g., sodium dodecyl sulfate(SDS), and the inclusion or exclusion of carrier DNA, are well known tothose skilled in the art. Various levels of stringency are accomplishedby combining these various conditions as needed. In a preferredembodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mMtrisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 MM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, Sequenase® (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE™ Amplification System(GIBCO BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) andthe ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

The nucleic acid sequences encoding HPPIP may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. Far example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrem, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PromoterFinder™ libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need lo screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO™ 4.06 Primer Analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., Genotyper™ and Sequence Navigator™, PerkinElmer), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HPPIP may be cloned in recombinant DNAmolecule s that direct expression of HPPIP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express HPPIP.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterHPPIP-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

In another embodiment, sequences encoding HPPIP may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, HPPIP itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide Synthesizer (Perkin Elmer).Additionally, the amino acid sequence of HPPIP, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman and Co., New York, N.Y.)

In order to express a biologically active HPPIP, the nucleotidesequences encoding HPPIP or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding HPPIP. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding HPPIP. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding HPPIP and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HPPIP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding HPPIP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding HPPIP. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding HPPIP can be achievedusing a multifunctional E. coli vector such as Bluescript® (Stratagene)or pSport1™ plasmid (GIBCO BRL). Ligation of sequences encoding HPPIPinto the vector's multiple cloning site disrupts the lacZ gene, allowinga colorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509.) When large quantities of HPPIP are needed,e.g. for the production of antibodies, vectors which direct high levelexpression of HPPIP may be used. For example, vectors containing thestrong, inducible T5 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of HPPIP. A numberof vectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia pastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, supra; and Grant et al. (1987) MethodsEnzymol. 153:516-54; Scorer, C. A. et al. (1994) Bio/Technology12:181-184.)

Plant systems may also be used for expression of HPPIP. Transcription ofsequences encoding HPPIP may be driven viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., Hobbs, S.or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York, N.Y.; pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences (encoding HPPIP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses HPPIP in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

For long term production of recombinant proteins in mammalian systems,stable expression of HPPIP in cell lines is preferred. For example,sequences encoding HPPIP can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apt⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; and Lowy, I. et al. (1980) Cell 22:81,7-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the immunoglycosides neomycinand G-418; and als or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additionalselectable genes have been described, e.g., trpB and hisD, which altercellular requirements for metabolites. (See, e.g., Hartman, S. C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech,Palo Alto, Calif.), β glucuronidase and its substrate β-D-glucuronoside,or luciferase and its substrate luciferin may be used. These markers canbe used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system. (See, e.g., Rhodes, C. A. et al. (1995) MethodsMol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHPPIP is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HPPIP can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HPPIP under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingHPPIP and that express HPPIP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression ofHPPIP using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes or HPPIP is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E.et al. (1997 and periodic supplements) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; andMaddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HPPIP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding HPPIP,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which naybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HPPIP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHPPIP may be designed to contain signal sequences which direct secretionof HPPIP through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HPPIP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric HPPIPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of HPPIP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the HPPIP encodingsequence and the heterologous protein sequence, so that HPPIP may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeledHPPIP may be achieved in vitro using the TNT™ rabbit reticulocyte lysateor wheat germ extract systems (Promega, Madison, Wis.). These systemscouple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

Fragments of HPPIP may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed bymanual techniques or by automation. Automated synthesis may be achieved,for example, using the Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer). Various fragments of HPPIP may be synthesized separatelyand then combined to produce the full length molecule.

THERAPEUTICS

Protein sequence analysis identifies HPPIP as human peptidyl-prolylisomerases. In addition, HPPIP is expressed in libraries from canceroustissues and from tissues involved in the immune response. Therefore,HPPIP appears to play a role in cancer and autoimmune/inflammatorydisorders. Therefore, in one embodiment, an antagonist of HPPIP may beadministered to a subject to treat or prevent a cancer. Such a cancermay include, but is not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds HPPIP may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express HPPIP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding HPPIP may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

In another embodiment, an antagonist of HPPIP may be administered to asubject to treat or prevent an autoimmune/inflammatory disorder. Such adisorder may include, but is not limited to, acquired immunodeficiencysyndrome (AIDS), Adcdison's disease, adult respiratory distresssyndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn'sdisease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythernatosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma. In one aspect, an antibody whichspecifically binds HPPIP may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express HPPIP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding HPPIP may be administered to a subject to treator prevent an autoimmune/inflammatory disorder including, but notlimited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate, agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HPPIP may be produced using methods which are generallyknown in the art. In particular, purified HPPIP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HPPIP. Antibodies to HPPIP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HPPIP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HPPIP have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HPPIP aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to HPPIP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “(chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HPPIP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HPPIP mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab)2 fragments. Alternatively, Fab expression librariesmay be constructed to allow rapid and easy identification of monoclonalFtb fragments with the desired specificity. (See, e.g., Huse, W. D. etal. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HPPIP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HPPIP epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingHPPIP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding HPPIP may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding HPPIP. Thus, complementary molecules orfragments may be used to modulate HPPIP activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding HPPIP.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding HPPIP. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

Genes encoding HPPIP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding HPPIP. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingHPPIP. Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHPPIP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences cf between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable.

The suitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HPPIP. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues ire availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cat;, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HPPIP,antibodies to HPPIP, and mimetics, agonists, antagonists, or inhibitorsof HPPIP. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and. administration may be foundin the latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HPPIP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HPPIP or fragments thereof, antibodies of HPPIP,and agonists, antagonists or inhibitors of HPPIP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index, and it can beexpressed as the ED₅₀/LD₅₀ ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used to formulate a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that includesthe ED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 Ig to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind HPPIP may beused for the diagnosis of disorders characterized by expression ofHPPIP, or in assays to monitor patients being treated with HPPIP oragonists, antagonists, or inhibitors o)f HPPIP. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for HPPIP include methodswhich utilize the antibody and a label to detect HPPIP in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring HPPIP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HPPIP expression. Normal or standard values for HPPIPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHPPIP under conditions suitable for complex formation The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of HPPIP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHPPIP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHPPIP may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of HPPIP, and tomonitor regulation of HPPIP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HPPIP or closely related molecules may be used to identifynucleic acid sequences which encode HPPIP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HPPIP, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theHPPIP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequences of SEQID NO:3, or SEQ ID NO:4, or from genomic sequences including promoters,enhancers, and introns of the HPPIP gene.

Means for producing specific hybridization probes for DNAs encodingHPPIP include the cloning of polynucleotide sequences encoding HPPIP orHPPIP derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HPPIP may be used for the diagnosis ofa disorder associated with expression of HPPIP. Examples of such adisorder include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus, and autoimmune/inflammatorydisorders such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma. Thepolynucleotide sequences encoding HPPIP may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patients to detect altered HPPIPexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding HPPIP may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHPPIP may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding HPPIP in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HPPIP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HPPIP, under conditions;suitable for hybridization or amplification. Standard hybridization maybe quantified by compiling the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HPPIP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HPPIP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HPPIP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HPPIPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P C. et al. (1993) J. Immunol.

Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem.229-236.) The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and at spectrophotometricor calorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingHPPIP may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HPPIP on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or aim of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carried, or affectedindividuals.

In another embodiment of the invention, HPPIP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenHPPIP and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with HPPIP, orfragments thereof, and washed. Bound HPPIP is then detected by methodswell known in the art. Purified HPPIP can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HPPIP specificallycompete with a test compound for binding HPPIP. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HPPIP.

In additional embodiments, the nucleotide sequences which encode HPPIPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. cDNA Library Construction

The BRAINON01 library is a normalized brain line library constructedusing 4.8×10⁶ independent clones from the BRAINOT03 brain library.Starting RNA was made from nontumorous brain tissue removed from a26-year old Caucasian male during cranioplasty and excision of acerebral meningeal lesion. Pathology for the associated tumor tissueindicated a grade 4 oligoastrocytoma in the right fronto-parietal partof the brain. Patient history included radiation therapy. The librarywas oligo(dT)-primed, and cDNAs were cloned directionally into thepSPORT1 vector using SalI (5′) and NotI (3′). The normalization andhybridization conditions were adapted from Soares et al. (Proc. Natl.Acad. Sci 91):9228-9232.) except that a significantly longer (48 hour)reannealing hybridization was used.

The TMLR3DT01 library was constructed using RNA isolated from peripheralblood mononuclear cells collected from two unrelated Caucasian maledonors (25 and 29 years old).

For library construction, the tissues were homogenized and lysed inTRIZOL™ reagent (1 gm tissue/10 ml TRIZOL™; GIBCO BRL), a monoplasticsolution of phenol and guanidine isothiocyanate, using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.Y.).After a brief incubation on ice, chloroform was added (1:5 v/v), and thelysate .was centrifuged. The upper chloroform layer was removed to afresh tube, and the RNA extracted with isopropanol, resuspended inDEPC-treated water, and DNase treated for 25 min at 37° C. The RNA wasre-extracted twice with acid phenol-chloroform pH 4.7 and precipitatedusing 0.3M sodium acetate and 2.5 volumes ethanol. The mRNA was isolatedwith the Qiagen Oligotex kit (QIAGEN, Inc., Chatsworth, Calif.) and usedto construct the cDNA library.

The mRNA was handled according to the recommended protocols in theSuperScript plasmid system (Cat. #18248-013, GIBCO BRL). cDNA synthesiswas initiated with a NotI-oligo d(T) primer. Double stranded cDNA wasblunted, ligated to EcoRI adaptors, digested with NotI, fractionated ona Sepharose CL4B column (Cat. #275105-01; Pharmacia), and those cDNAsexceeding 400 bp were ligated into the NotI and EcoRI sites of eitherthe pSPORT1 (BRAINON01) or Lambda UniZAP (TMLR3DT01) vector. Theresulting plasmids were subsequently transformed into DH5α™ competentcells (Cat. #18258-012; GIBCO BRL).

II. Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 plasmid kit (QIAGEN Inc). The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (GIBCO BRL) with carbenicillin at 25 mg/L andglycerol at 0.4%; 2) after inoculation, the cultures were incubated for19 hours and then the cells were lysed with 0.3 ml of lysis buffer; and3) following isopropanol precipitation, the plasmid DNA pellets wereresuspended in 0.1 ml of distilled water. The plasmid DNA samples werestored at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems; andthe reading frame was determined.

III. Similarity Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofsimilarity using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms couldhave been used when dealing with primary sequence patterns and secondarystructure gap penalties. (See, e.g., Smith, T. et al. (1992) ProteinEngineering 5:35-51.) The sequences disclosed in this application havelengths of at least 49 nucleotides and have no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for similarity.

Additionally, sequences identified from cDNA libraries may be analyzedto identify those gene sequences encoding conserved protein motifs usingan appropriate analysis program, e.g., BLOCKS. BLOCKS is a weightedmatrix analysis algorithm based on short amino acid segments, or blocks,compiled from the PROSITE database. (Bairoch, A. et al. (1997) NucleicAcids Res. 25:217-221.) The BLOCKS algorithm is useful for classifyinggenes with unknown functions. (Henikoff S. And Henikoff G. J., NucleicAcids Research (1991) 19:6565-6572.) Blocks, which are 3-60 amino acidsin length, correspond to the most highly conserved regions of proteins.The BLOCKS algorithm compares a query sequence with a weighted scoringmatrix of blocks in the BLOCKS database. Blocks in the BLOCKS databaseare calibrated against protein sequences with known functions from theSWISS-PROT database to determine the stochastic distribution of matches.Similar databases such as PRINTS, a protein fingerprint database, arealso searchable using the BLOCKS algorithm. (Attwood, T. K. et al.(1997) J. Chem. Inf. Comput. Sci. 37:417-424.) PRINTS is based onnon-redundant sequences obtained from sources such as SWISS-PROT,GenBank, PIR, and NRL-3D.

The BLOCKS algorithm searches for matches between a query sequence andthe BLOCKS or PRINTS database and evaluates the statistical significanceof any matches found. Matches from a BLOCKS or PRINTS search can beevaluated on two levels, local similarity and global similarity. Thedegree of local similarity is measured by scores, and the extent ofglobal similarity is measured by score ranking and probability values isscore of 1000 or greater for a BLOCKS match of highest ranking indicatesthat the match falls within the 0.5 percentile level of false positiveswhen the matched block is calibrated against SWISS-PROT. Likewise, aprobability value of less than 1.0×10⁻³ indicates that the match wouldoccur by chance no more than one time in every 1000 searches. Only thosematches with a cutoff score of 1000 or greater and a cutoff probabilityvalue of 1.0×10⁻³ or less are considered in the functional analyses ofthe protein sequences in the Sequence Listing.

Nucleic and amino acid sequences of the Sequence Listing may also beanalyzed using PFAM. PFAM is a Hidden Markov Model (HMM) based protocoluseful in protein family searching. HMM is a probabilistic approachwhich analyzes consensus primary structures of gene families. (See,e.g., Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365.)

The PFAM database contains protein sequences of 527 protein familiesgathered from publicly available sources, e.g., SWISS-PROT and PROSITE.PFAM searches for well characterized protein domain families using twohigh-quality alignment routines, seed alignment and full alignment.(See, e.g., Sonnhammer, E. L. L. et al. (1997) Proteins 28:405-420.) Theseed alignment utilizes the hmmls program, a program that searches forlocal matches, and a non-redundant set of the PFAM database. The fullalignment utilizes the hmmfs program, a program that searches formultiple fragments in long sequences, e.g., repeats and motifs, and allsequences in the PFAM database. A result or score of 100 “bits” cansignify that it is 2¹⁰⁰-fold more likely that the sequence is a truematch to the model or comparison sequence. Cutoff scores which rangefrom 10 to 50 bits are generally used for individual protein familiesusing the SWISS-PROT sequences as model or comparison sequences.

Two other algorithms, SIGPEPT and TM, both based on the HMM algorithmdescribed above (see, e.g., Eddy, supra; and Sonnhammer, supra),identify potential signal sequences and transmembrane domains,respectively. SIGPEPT was created using protein sequences having signalsequence annotations derived from SWISS-PROT. It contains about 1413non-redundant signal sequences ranging in length from 14 to 36 aminoacid residues. TM was created similarly using transmembrane domainannotations. It contains about 453 non-redundant transmembrane sequencesencompassing 1579 transmembrane domain segments. Suitable HMM modelswere constructed using the above sequences and were refined with knownSWISS-PROT signal peptide sequences or transmembrane domain sequencesuntil a high correlation coefficient, a measurement of the correctnessof the analysis, was obtained. Using the protein sequences from theSWISS-PROT database as a test set, a cutoff score of 11 bits, asdetermined above, correlated with 91-94% true-positives and about 4. 1%false-positives, yielding a correlation coefficient of about 0.87-0.90for SIGPEPT. A score of 11 bits for TM will typically give the followingresults: 75% true positives; 1.72% false positives; and a correlationcoefficient of 0.76. Each search evaluates the statistical significanceof any matches found and reports only those matches that score at least11 bits.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect: the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ™ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or similar.

The basis of the search is the product score, which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact. Similarmolecules are usually identified by selecting those which show productscores between 15 and 40, although lower scores may identify relatedmolecules.

The results of Northern analysis are reported as a list of libraries inwhich the transcript encoding HPPIP occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of HPPIP Encoding Polynucleotides

The nucleic acid sequences of Incyte Clones 2291164 and 289973 were usedto design oligonucleotide primers for extending partial nucleotidesequences to full length. For each nucleic acid sequence, one primer wassynthesized to initiate extension of an antisense polynucleotide, andthe other was synthesized to initiate extension of a sensepolynucleotide. Primers were used to facilitate the extension of theknown sequence “outward” generating amplicons containing new unknownnucleotide sequence for the region of interest. The initial primers weredesigned from the CDNA using OLIGO™ 4.06 (National Biosciences,Plymouth, Minn.), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer—primer dimerizations was avoided.

Selected human cDNA libraries (GIBCO BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR™ kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier Thermal Cycler(PTC200; M.J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all, other componentsof the kit, with the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for anadditional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 minStep 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for anadditional 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (andholding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK™ (QIAGEN Inc.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:3 and SEQ ID NO:4are used to obtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:3 and SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although thelabeling-, of oligonucleotides, consisting of about 20 base pairs, isspecifically described, essentially the same procedure is used withlarger nucleotide fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO™ 4.06 software (NationalBiosciences) and label(ed by combining 50 pmol of each oligomer, 250 μCiof [γ-³²P] adenosine triphosphate (Amersham, Chicago, Ill.), and T4polynucleotide kinase (DuPont NEN®, Boston, Mass.). The labeledoligonucleotides are substantially purified using a Sephadex™ G-25superfine size exclusion dextran bead column (Pharmacia & Upjohn,Kalamazoo, Mich.). An aliquot containing 10⁷ counts per minute of thelabeled probe is used in a typical membrane-based hybridization analysisof human genomic DNA digested with one of the following endonucleases:Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu nI (DuPont NEN, Boston,Mass.).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots to film for severalhours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE™. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; andShalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the HPPIP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HPPIP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO™ 4.06 software andthe coding sequence of HPPIP. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HPPIP-encoding transcript.

IX. Expression of HPPIP

Expression and purification of HPPIP is achieved using bacterial orvirus-based expression systems. For expression of HPPIP in bacteria,CDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express HPPIP uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof HPPIP in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with CDNAencoding HPPIP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, HPPIP is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma iaponicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (Pharmacia,Piscataway, N.J.). Following purification, the GST moiety can beproteolytically cleaved from HPPIP at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutivehistidine residues, enables purification on metal-chelate resins (QIAGENInc, Chatsworth, Calif.). Methods for protein expression andpurification are discussed in Ausubel, F.

M. et al. (1995 and periodic supplements) Current Protocols in MolecularBiology, John Wiley & Sons, New York, NY, ch 10, 16. Purified HPPIPobtained by these methods can be used directly in the following activityassay.

X. Demonstration of HPPIP Activity

Peptidyl prolyl cis/trans isomerase activity of HPPIP can be assayed byan enzyme assay described by Rahfeld, J. U., et al. (1994) (FEBS Lett.352: 180-184). The assay is performed at 10° C. in 35 mM HEPES buffer,pH 7.8, containing chymotrypsin (0.5 mg/ml) and HPPIP at a variety ofconcentrations. Under these assay conditions, the substrate,Suc-Ala-Xaa-Pro-Phe-4-NA, is in equilibrium with respect to the prolylbond, with 80-95% in trans and 5-20% in cis conformation. An aliquot (2ul) of the substrate dissolved in dimethyl sulfoxide (10 mg/ml) is addedto the reaction mixture described above. The trans to cis conversion ismeasured by the hydrolysis of the cis conformer by chymotrypsin,producing 4-nitroanilide which is detected spectrophotometrically byit's absorbance at 390 nm. 4-Nitroanilide appears in a time-dependentand an HPPIP concentration-dependent manner.

XI. Functional Assays

HPPIP function is assessed by expressing the sequences encoding HPPIP atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPORT™ (Life Technologies, Gaithersburg, Md.) and pCR™ 3.1(Invitrogen, Carlsbad, Calif., both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, preferably of -endothelial or hematopoietic origin,using either liposome formulations or electroporation. 1-2 μg of anadditional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP)(Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flowcytometry (FCM), an automated, laser optics-based technique, is used toidentify transfected cells expressing GFP or CD64-GFP, and to evaluateproperties, for example, their apoptotic state. FCM detects andquantifies the uptake of fluorescent molecules that diagnose eventspreceding or coincident with cell death. These events include changes innuclear DNA content as measured by staining of DNA with propidiumiodide; changes in cell size and granularity as measured by forwardlight scatter and 90 degree side light scatter; down-regulation of DNAsynthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins asmeasured by reactivity with specific antibodies; and alterations inplasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry,Oxford, New York, N.Y.

The influence of HPPIP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding HPPIPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding HPPIP and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

XII. Production of HPPIP Specific Antibodies

HPPIP substantially purified using polyacrylamide gel electrophoresis(PAGE)(see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the HPPIP amino acid sequence is analyzed usingLASERGENE™ software (DNASTAR Inc.) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel supra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anApplied Biosystems Peptide Synthesizer Model 43 1A using fmoc-chemistryand coupled to KLH (Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

XIII. Purification of Naturally Occurring HPPIP Using SpecificAntibodies

Naturally occurring or recombinant HPPIP is substantially purified byimmunoaffinity chromatography using antibodies specific for HPPIP. Animmunoaffinity column is constructed by covalently coupling anti-HPPIPantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HPPIP are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of HPPIP (e.g., high ionic strength buffers in the presenceof detergent). The column is elited under conditions that disruptantibody/HPPIP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andHPPIP is collected.

XIV. Identification of Molecules Which Interact with HPPIP

HPPIP, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HPPIP, washed, and anywells with labeled HPPIP complex are assayed. Data obtained usingdifferent concentrations of HPPIP are used to calculate values for thenumber, affinity, and association of HPPIP with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

4 472 amino acids amino acid single linear unknown BRAINON01 2291164 1Met Ser Asn Ile Tyr Ile Gln Glu Pro Pro Thr Asn Gly Lys Val Leu 1 5 1015 Leu Lys Thr Thr Ala Gly Asp Ile Asp Ile Glu Leu Trp Ser Lys Glu 20 2530 Ala Pro Lys Ala Cys Arg Asn Phe Ile Gln Leu Cys Leu Glu Ala Tyr 35 4045 Tyr Asp Asn Thr Ile Phe His Arg Val Val Pro Gly Phe Ile Val Gln 50 5560 Gly Gly Asp Pro Thr Gly Thr Gly Ser Gly Gly Glu Ser Ile Tyr Gly 65 7075 80 Ala Pro Phe Lys Asp Glu Phe His Ser Arg Leu Arg Phe Asn Arg Arg 8590 95 Gly Leu Val Ala Met Ala Asn Ala Gly Ser His Asp Asn Gly Ser Gln100 105 110 Phe Phe Phe Thr Leu Gly Arg Ala Asp Glu Leu Asn Asn Lys HisThr 115 120 125 Ile Phe Gly Lys Val Thr Gly Asp Thr Val Tyr Asn Met LeuArg Leu 130 135 140 Ser Glu Val Asp Ile Asp Asp Asp Glu Arg Pro His AsnPro His Lys 145 150 155 160 Ile Lys Ser Cys Glu Val Leu Phe Asn Pro PheAsp Asp Ile Ile Pro 165 170 175 Arg Glu Ile Lys Arg Leu Lys Lys Glu LysPro Glu Glu Glu Val Lys 180 185 190 Lys Leu Lys Pro Lys Gly Thr Lys AsnPhe Ser Leu Leu Ser Phe Gly 195 200 205 Glu Glu Ala Glu Glu Glu Glu GluGlu Val Asn Arg Val Ser Gln Ser 210 215 220 Met Lys Gly Lys Ser Lys SerSer His Asp Leu Leu Lys Asp Asp Pro 225 230 235 240 His Leu Ser Ser ValPro Val Val Glu Ser Glu Lys Gly Asp Ala Pro 245 250 255 Asp Leu Val AspAsp Gly Glu Asp Glu Ser Ala Glu His Asp Glu Tyr 260 265 270 Ile Asp GlyAsp Glu Lys Asn Leu Met Arg Glu Arg Ile Ala Lys Lys 275 280 285 Leu LysLys Asp Thr Ser Ala Asn Val Lys Ser Ala Gly Glu Gly Glu 290 295 300 ValGlu Lys Lys Ser Val Ser Arg Ser Glu Glu Leu Arg Lys Glu Ala 305 310 315320 Arg Gln Leu Lys Arg Glu Leu Leu Ala Ala Lys Gln Lys Lys Val Glu 325330 335 Asn Ala Ala Lys Gln Ala Glu Lys Arg Ser Glu Glu Glu Glu Ala Pro340 345 350 Pro Asp Gly Ala Val Ala Glu Tyr Arg Arg Glu Lys Gln Lys TyrGlu 355 360 365 Ala Leu Arg Lys Gln Gln Ser Lys Lys Gly Thr Ser Arg GluAsp Gln 370 375 380 Thr Leu Ala Leu Leu Asn Gln Phe Lys Ser Lys Leu ThrGln Ala Ile 385 390 395 400 Ala Glu Thr Pro Glu Asn Asp Ile Pro Glu ThrGlu Val Glu Asp Asp 405 410 415 Glu Gly Trp Met Ser His Val Leu Gln PheGlu Asp Lys Ser Arg Lys 420 425 430 Val Lys Asp Ala Ser Met Gln Asp SerAsp Thr Phe Glu Ile Tyr Asp 435 440 445 Pro Arg Asn Pro Val Asn Lys ArgArg Arg Glu Glu Ser Lys Lys Leu 450 455 460 Met Arg Glu Lys Lys Glu ArgArg 465 470 443 amino acids amino acid single linear unknown TMLR3DT01289973 2 Met Gly Ser Ala Phe Glu Arg Val Val Arg Arg Val Val Gln Glu Leu1 5 10 15 Asp His Gly Gly Glu Phe Ile Pro Val Thr Ser Leu Gln Ser SerThr 20 25 30 Gly Phe Gln Pro Tyr Cys Leu Val Val Arg Lys Pro Ser Ser SerTrp 35 40 45 Phe Trp Lys Pro Arg Tyr Lys Cys Val Asn Leu Ser Ile Lys AspIle 50 55 60 Pro Asp Ala Ala Glu Pro Asp Val Gln Arg Gly Arg Ser Phe HisPhe 65 70 75 80 Tyr Asp Ala Met Asp Gly Gln Ile Gln Gly Ser Val Glu LeuAla Ala 85 90 95 Pro Gly Gln Ala Lys Ile Ala Gly Gly Ala Ala Val Ser AspSer Ser 100 105 110 Ser Thr Ser Met Asn Val Tyr Ser Leu Ser Val Asp ProAsn Thr Trp 115 120 125 Gln Thr Leu Leu His Glu Arg His Leu Arg Gln ProGlu His Lys Val 130 135 140 Leu Gln Gln Leu Arg Thr Arg Gly Asp Asn ValTyr Val Val Thr Glu 145 150 155 160 Val Leu Gln Thr Gln Lys Glu Val GluVal Thr Arg Thr His Lys Arg 165 170 175 Glu Gly Ser Gly Arg Phe Ser LeuPro Gly Ala Thr Cys Leu Gln Gly 180 185 190 Glu Gly Gln Gly His Leu SerGln Lys Lys Thr Val Thr Ile Pro Ser 195 200 205 Gly Ser Thr Leu Ala PheArg Val Ala Gln Leu Val Ile Asp Ser Asp 210 215 220 Leu Asp Val Leu LeuPhe Pro Asp Lys Lys Gln Arg Thr Phe Gln Pro 225 230 235 240 Pro Ala ThrGly His Lys Arg Ser Thr Ser Glu Gly Ala Trp Pro Gln 245 250 255 Leu ProSer Gly Leu Ser Met Met Arg Cys Leu His Asn Phe Leu Thr 260 265 270 AspGly Val Pro Ala Glu Gly Ala Phe Thr Glu Asp Phe Gln Gly Leu 275 280 285Arg Ala Glu Val Glu Thr Ile Ser Lys Glu Leu Glu Leu Leu Asp Arg 290 295300 Glu Leu Cys Gln Leu Leu Leu Glu Gly Leu Glu Gly Val Leu Arg Asp 305310 315 320 Gln Leu Ala Leu Arg Ala Leu Glu Glu Ala Leu Glu Gln Gly GlnSer 325 330 335 Leu Gly Pro Val Glu Pro Leu Asp Gly Pro Ala Gly Ala ValLeu Glu 340 345 350 Cys Leu Val Leu Ser Ser Gly Met Leu Val Pro Glu LeuAla Ile Pro 355 360 365 Val Val Tyr Leu Leu Gly Ala Leu Thr Met Leu SerGlu Thr Arg Ala 370 375 380 Gln Ala Ala Gly Gly Gly Ala Gly Val Ala GluLeu Leu Gly Pro Leu 385 390 395 400 Glu Leu Val Gly Ser Leu Leu Glu GlnSer Arg Arg Ser Ala His His 405 410 415 Val Pro His Pro Gly Leu Met TrpAsn Thr Gly Arg Lys Arg Pro Ala 420 425 430 Cys Val Cys Trp Thr Val TrpLeu Asp Trp Gly 435 440 2105 base pairs nucleic acid single linearunknown BRAINON01 2291164 3 CCGGTAACAA CATGGCGGCG TCCGTGAGGG GCTCCTTTGGGCAGGGGTAG TGTTTGGTGT 60 CCCTGTCTTG CGTGATATTG ACAAACTGAA GCTTTCCTGCACCACTGGAC TTAAGGAAGA 120 GTGTACTCGT AGGCGGACAG CTTTAGTGGC CGGCCGGCCGCTCTCATCCC CCGTAAGGAG 180 CAGAGTCCTT TGTACTGACC AAGATGAGCA ACATCTACATCCAGGAGCCT CCCACGAATG 240 GGAAGGTTTT ATTGAAAACT ACAGCTGGAG ATATTGACATAGAGTTGTGG TCCAAAGAAG 300 CTCCTAAAGC TTGCAGAAAT TTTATCCAAC TTTGTTTGGAAGCTTATTAT GACAATACCA 360 TTTTTCATAG AGTTGTGCCT GGTTTCATAG TCCAAGGCGGAGATCCTACT GGCACAGGGA 420 GTGGTGGAGA GTCTATCTAT GGAGCGCCAT TCAAAGATGAATTTCATTCA CGGTTGCGTT 480 TTAATCGGAG AGGACTGGTT GCCATGGCAA ATGCTGGTTCTCATGATAAT GGCAGCCAGT 540 TTTTCTTCAC ACTGGGTCGA GCAGATGAAC TTAACAATAAGCATACCATC TTTGGAAAGG 600 TTACAGGGGA TACAGTATAT AACATGTTGC GACTGTCAGAAGTAGACATT GATGATGACG 660 AAAGACCACA TAATCCACAC AAAATAAAAA GCTGTGAGGTTTTGTTTAAT CCTTTTGATG 720 ACATCATTCC AAGGGAAATT AAAAGGCTGA AAAAAGAGAAACCAGAGGAG GAAGTAAAGA 780 AATTGAAACC CAAAGGCACA AAAAATTTTA GTTTACTTTCATTTGGAGAG GAAGCTGAGG 840 AAGAAGAGGA GGAAGTAAAT CGAGTTAGTC AGAGCATGAAGGGCAAAAGC AAAAGTAGTC 900 ATGACTTGCT TAAGGATGAT CCACATCTCA GTTCTGTTCCAGTTGTAGAA AGTGAAAAAG 960 GTGATGCACC AGATTTAGTT GATGATGGAG AAGATGAAAGTGCAGAGCAT GATGAATATA 1020 TTGATGGTGA TGAAAAGAAC CTGATGAGAG AAAGAATTGCCAAAAAATTA AAAAAGGACA 1080 CAAGTGCGAA TGTTAAATCA GCTGGAGAAG GAGAAGTGGAGAAGAAATCA GTCAGCCGCA 1140 GTGAAGAGCT CAGAAAAGAA GCAAGACAAT TAAAACGGGAACTCTTAGCA GCAAAACAAA 1200 AAAAAGTAGA AAATGCAGCA AAACAAGCAG AAAAAAGAAGTGAAGAGGAA GAAGCCCCTC 1260 CAGATGGTGC TGTTGCCGAA TACAGAAGAG AAAAGCAAAAGTATGAAGCT TTGAGGAAGC 1320 AACAGTCAAA GAAGGGAACT TCCCGGGAAG ATCAGACCCTTGCACTGCTG AACCAGTTTA 1380 AATCTAAACT CACTCAAGCA ATTGCTGAAA CGCCTGAAAATGACATTCCT GAAACAGAAG 1440 TAGAAGATGA TGAAGGATGG ATGTCACATG TACTTCAGTTTGAGGATAAA AGCAGAAAAG 1500 TGAAAGATGC AAGCATGCAA GACTCAGATA CATTTGAAATCTATGATCCT CGGAATCCAG 1560 TGAATAAAAG AAGGAGGGAA GAAAGCAAAA AGCTGATGAGAGAGAAAAAA GAAAGAAGAT 1620 AAAATGAGAA TAATGATAAC CAGAACTTGC TGGAAATGTGCCTACAATGG CCTTGTAACA 1680 GCCATTGTTC CCAACAGCAT CACTTAGGGG TGTGAAAAGAAGTATTTTTG AACCTGTTGT 1740 CTGGTTTTGA AAAACAATTA TCTTGTTTTG CAAATTGTGGAATGATGTAA GCAAATGCTT 1800 TTGGTTACTG GTACATGTGT TTTTTCCTAG CTGACCTTTTATATTGCTAA ATCTGAAATA 1860 AAATAACTTT CCTTCCACAT TACATGTTAA CCATTGCAGACTGCAAGCCT GTTTGTGTCC 1920 TTTTACCCTA AAATATATGA AGGCTTCCTT TTCAAGATTTTTTTATAAGA AGTTCCTACA 1980 GAAAGAATAT TTGTGGGAAA CCTCCCTTTC ACTAACTTCAGAATATAATA AAATATTATA 2040 AATAAAATAT AGAATATAAA TATGGATGGG GTTTTTTGCATATAAATATT TCAAAATATC 2100 CAAGC 2105 1449 base pairs nucleic acidsingle linear unknown TMLR3DT01 289973 4 CCAGCTCCTG CTCGCCGGACGGCTCCCAGG GAGAGCAGAC GCGCCAGACG CGCCACCCTC 60 GGGGCGCCGA CGGTCACGGAGCATGGGGTC GGCCTTTGAG CGGGTAGTCC GGAGAGTGGT 120 CCAGGAGCTG GACCATGGTGGGGAGTTCAT CCCTGTGACC AGCCTGCAGA GCTCCACTGG 180 CTTCCAGCCC TACTGCCTGGTGGTTAGGAA GCCCTCAAGC TCATGGTTCT GGAAACCCCG 240 TTATAAGTGT GTCAACCTGTCTATCAAGGA CATCCTGGAG CCGGATGCCG CGGAACCAGA 300 CGTGCAGCGT GGCAGGAGCTTCCACTTCTA CGATGCCATG GATGGGCAGA TACAGGGCAG 360 CGTGGAGCTG GCAGCCCCAGGACAGGCAAA GATCGCAGGC GGGGCCGCGG TGTCTGACAG 420 CTCCAGCACC TCAATGAATGTGTACTCGCT GAGTGTGGAC CCTAACACCT GGCAGACTCT 480 GCTCCATGAG AGGCACCTGCGGCAGCCAGA ACACAAAGTC CTGCAGCAGC TGCGCACGCG 540 CGGGGACAAC GTGTACGTGGTGACTGAGGT GCTGCAGACA CAGAAGGAGG TGGAAGTCAC 600 GCGCACCCAC AAGCGGGAGGGCTCGGGCCG GTTTTCCCTG CCCGGAGCCA CGTGCTTGCA 660 GGGTGAGGGC CAGGGCCATCTGAGCCAGAA GAAGACGGTC ACCATCCCCT CAGGCAGCAC 720 CCTCGCATTC CGGGTGGCCCAGCTGGTTAT TGACTCTGAC TTGGACGTCC TTCTCTTCCC 780 GGATAAGAAG CAGAGGACCTTCCAGCCACC CGCGACAGGC CACAAGCGTT CCACGAGCGA 840 AGGCGCCTGG CCACAGCTGCCCTCTGGCCT CTCCATGATG AGGTGCCTCC ACAACTTCCT 900 GACAGATGGG GTCCCTGCGGAGGGGGCGTT CACTGAAGAC TTCCAGGGCC TACGGGCAGA 960 GGTGGAGACC ATCTCCAAGGAACTGGAGCT TTTGGACAGA GAGCTGTGCC AGCTGCTGCT 1020 GGAGGGCCTG GAGGGGGTGCTGCGGGACCA GCTGGCCCTG CGAGCCTTGG AGGAGGCGCT 1080 GGAGCAGGGC CAGAGCCTTGGGCCGGTGGA GCCCCTGGAC GGTCCAGCAG GTGCTGTCCT 1140 GGAGTGCCTG GTGTTGTCCTCCGGAATGCT GGTGCCGGAA CTCGCTATCC CTGTTGTCTA 1200 CCTGCTGGGG GCACTGACCATGCTGAGTGA AACGCGAGCA CAAGCTGCTG GCGGAGGCGC 1260 TGGAGTCGCA GAACTGTTGGGGCCGCTCGA GCTGGTGGGC AGCCTCTTGG AGCAGAGTGC 1320 CCGTGGCAGG AGCGCACACCATGTCCCTCA CCCCGGGCTC ATGTGGAACA CTGGGCGCAA 1380 GAGACCGGCC TGTGTTTGCTGGACGGTGTG GCTAGACTGG GGATGACATC CCACGTGTGT 1440 GGGAACGAC 1449

What is claimed is:
 1. An isolated polynucleotide encoding an amino acidsequence selected from the group consisting of: SEQ ID NO:1, and SEQ IDNO:2.
 2. An isolated polynucleotide encoding a polypeptide selected fromthe group consisting of: (a) a polypeptide comprising an amino acidsequences having ail least 90% amino acid sequence identity with theamino acid sequence as shown in SEQ ID NO 1, and (b) a polypeptidecomprising an amino acid sequence having at least 90% amino acidsequence identity with the amino acid sequence as shown in SEQ ID NO 2,wherein the polypeptide demonstrates human peptidyl-prolyl isomeraseactivity.
 3. An isolated polynucleotide of claim 2 encoding apolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO 1, and(b) the amino acid sequence as shown in SEQ ID NO
 2. 4. An isolatedpolynucleotide encoding an enzymatically active fragment of apolypeptide, the polypeptide selected from the group consisting of: (a)a polypeptide having an amino acid sequence as shown in SEQ ID NO 1, and(b) a polypeptide having an amino acid sequence as shown in SEQ ID NO 2.5. An isolated polynucleotide having a sequence which is complementaryto the polynucleotide of claim
 2. 6. An isolated polynucleotide having asequence which is complementary to the polynucleotide of claim
 3. 7. Anisolated polynucleotide having a sequence which is complementary to thepolynucleotide of claim
 4. 8. An isolated polynucleotide comprising apolynucleotide sequence selected from the group consisting of: (a) apolynucleotide sequence having at least 70% nucleotide sequence identitywith SEQ ID NO 3, and (b) a polynucleotide sequence having at least 70%nucleotide sequence identity with SEQ ID NO
 4. 9. An isolatedpolynucleotide of claim 8, selected from the group consisting of: (a)SEQ ID NO 3, and (b) SEQ ID NO
 4. 10. An isolated polynucleotide havinga sequence which is complementary to the polynucleotide of claim
 8. 11.An isolated polynucleotide having a sequence which is complementary tothe polynucleotide of claim
 9. 12. An expression vector comprising thepolynucleotide of claim 8 operably linked to a promoter.
 13. A host cellcomprising the expression vector of claim
 12. 14. A method for producinga polypeptide, the method comprising the steps of: a) culturing the hostcell of claim 13 under conditions suitable for the expression of thepolypeptide; and b) recovering the polypeptide from the host cellculture.